The ultimate goal of this project is to develop a safe and effective topical vaccine against the dengue virus (DENV). DENV causes an estimated 30 to 50 million cases of debilitating fever leading to over 20,000 deaths worldwide every year. DNA-based vaccines have great potential against DENV because they can more quickly generate a balanced immune response, are less expensive to produce, and have greater temperature stability than current DENV vaccine candidates in clinical trial. However, DNA vaccines have historically suffered from low immunogenicity. The vaccine that we propose is engineered to overcome this barrier-to- success in three key ways. First, the US Centers for Disease Control and Prevention (CDC) vaccine incorporates advances that redirect humoral immunity away from the production of non-protective and potentially pathogenic antibodies to increase the production of potently neutralizing and protective antibodies. Second, the vaccine is delivered directly to the network of immune effector cells in the skin via GeneSegues' (GSI's) topically administered sub-50 nanometer (s50) capsules. Third, the s50 encapsulated vaccine exploits the efficient, size-sensitive lipid raft uptake pathway to traffic directly to the nucleusof immune effector cells, addressing a major hurdle to DNA delivery. In this Phase 1 study, we propose to develop a topical DENV DNA vaccine by focusing on serotype-2 (DENV-2), with four specific aims. First, we will build upon pilot in vivo s50 DENV-2 vaccine delivery studies to determine optimal topical delivery site parameters in a mouse model. Second, for the selected delivery and application site, we will mechanistically assess differential adjuvants and dosing parameters, by comparing early percent effector cell transfection and antigen expression with subsequent neutralizing antibody response and persistence. Third, using the two best s50 vaccine candidates identified in Aims 1 and 2, we will characterize cell-mediated immune responses by examining the establishment and persistence of DENV-2 specific immunological memory. Fourth, in a separate study arm, we will compound the s50 DENV-2 DNA vaccine in a panel of semisolid vehicles (lotions and/or gels) to obtain maximum and uniform dose delivery, and execute a proof-of-principle study in mice with the best compounded candidate, with a key goal of achieving equivalent or superior protective neutralizing antibody titers vs. naked DNA delivered via electroporation and via intramuscular injection. Future work will expand and transition to the three remaining dengue serotypes (DENV-1, -3 and -4) and determine the optimum formulation to elicit tetravalent balanced, protective, and long lasting immunity within two to three months. Vaccine efficacy and safety will be assessed by using the DENV vaccine and disease AG129 mouse model, including lethal challenge and sublethal antibody-dependent enhancement of infection studies. We will also conduct manufacturing scale-up and other tasks necessary to progress to IND submission and human clinical trials.